The recent explosion of genomic data has offered an unprecedented opportunity to study the molecular basis for developmental and disease processes. Although microarray techniques for profiling the genes expressed in a given tissue have become commonplace, methods for determining the exact spatial and temporal extent of gene expression are still in need of improvement. In situ hybridization techniques for gene expression studies often have limited sensitivity and significant background signal even in locations where no target genes are present. Furthermore, existing technologies do not fully exploit the potential for imaging many genes simultaneously. The goal of the proposed research is to adapt a newly developed nanosensor technology for multiplexed in situ amplification of gene expression. This amplification tool, termed hybridization chain reaction (HCR), reduces background by activating only when a probe molecule binds specifically to its target. This event triggers the self-assembly of a tethered 'polymer' from fluorescently-labeled DMA hairpins. Parallel multiplexing can be achieved simply by using independent HCR amplifiers for each unique target species. The research plan involves the design, validation and application of in situ HCR amplifiers. Specific aims are: 1: Design triggered, multiplexed, nonlinear HCR amplifiers, and refine the computational tools for the design of HCR amplifiers. 2: Validate the spatial localization, sensitivity, specificity and multiplexing of the amplifiers in situ using nanolithography techniques to precisely pattern target molecules on surfaces. 3: Apply HCR to in situ hybridization of patterning genes in avian embryos, testing for the co- expression of genetic markers predicted by previous studies. The objective is to develop in situ HCR amplifiers that will enable the sensitive and simultaneous detection of numerous targets in biological specimens. If successful, this nanotechnology will serve as an important adjunct to modern genomic and proteomic tools in settings ranging from biological experiments to tissue biopsies.